Genetic structure of Ponto‐Caspian trout populations shows gene flow among river drainages and supports resident Salmo rizeensis as a genetically distinct taxon

Abstract To assess the genetic structure of Ponto‐Caspian brown trout (Salmo trutta complex) populations, we analyzed both mitochondrial DNA sequences and genotypes at 10 microsatellite loci of fish caught in the Black Sea and from nine river catchments in Georgia, flowing into either the Black or Caspian seas. The results show that: (1) there is substantial genetic differentiation among Ponto‐Caspian trout populations, both among the populations of different nominal species and within those of the same species; (2) the genetic distance between conspecific populations from the Black and Caspian Sea basins exceeds that among the populations within the same basin. Moreover, within drainages, genetic distance correlates with the geographic distance; (3) the Black Sea itself is not a barrier to gene flow among the watersheds draining into the Black Sea; (4) some populations in the headwaters of the rivers draining into the Black Sea Basin fall out of this pattern and likely form a separate, non‐anadromous (resident) taxon, previously described from northeastern Turkey as Salmo rizeensis. This hypothesis is supported by mitochondrial DNA phylogeny. The presence of both anadromous and resident populations in a single river basin calls for a substantial re‐thinking of speciation patterns and taxonomy of Eurasian brown trout.

as S. trutta (Bernatchez, 2001). S. trutta fario was the name used for the resident form of brown trout, irrespective of geographic origin (Jonsson & Jonsson, 2011).
In more recent decades, however, ichthyologists recognized a number of trout populations from Southern Europe, West Asia, and North Africa as separate species, distinct from the Central-and Northern European S. trutta (Kottelat & Freyhof, 2007;Segherloo et al., 2021;Turan et al., 2009). This number has increased to as many as 47 extant nominal species, and most of these taxa exhibit some level of genetic differentiation (Segherloo et al., 2021).
Within the Ponto-Caspian area, 14 species of brown trout are formally described (Kottelat & Freyhof, 2007;Ninua et al., 2018;Segherloo et al., 2021;Turan et al., 2009Turan et al., , 2022, and two more taxa considered to be distinct, although non-named species. However, validity of some of those, including Salmo ciscaucasicus from the Caspian Sea basin, as well as Salmo coruhensis and Salmo rizeensis from the Black Sea basin, was put under question (Segherloo et al., 2021; Table 1). Ninua et al. (2018) investigated the differentiation of trout from rivers from both Black and Caspian Sea drainages, and the Black Sea coastal area of Georgia, including in their analysis published mitochondrial cytochrome b and control region DNA sequences of brown trout from the Ponto-Caspian area. They synonymized S. coruhensis from the southern Black Sea drainage and S. trutta from Danube drainage with Salmo labrax, but did not question separate species' status for a non-anadromous resident form, S. rizeensis (Turan et al., 2009). Ninua et al. (2018) also posit two species, Salmo caspius, and S. ciscaucasicus, in the rivers flowing into the Caspian Sea. Besides, they showed the monophyletic origin of all brown trout taxa from the Black, Caspian, and Aral Seas (Ponto-Caspian basin), distinct from related fish from the Mediterranean, Persian Gulf, and Atlantic drainages. This interpretation was later supported by the genomic data of Segherloo et al. (2021).
The existing taxonomy reflects overall genetic differentiation among distinct geographic populations; however, it is unclear how the distribution of genetic variation and gene flow is partitioned among taxa, and whether differentiation among local trout populations scales with geographic distance, irrespective of formal taxonomy.
Moreover, neither of the recent studies, except Turan et al. (2009), and the very old ecological observations of Barach (1962), addresses the genetic differentiation of anadromous versus resident populations. This is an important question: it is not entirely clear whether the anadromous life history is an inherent feature of certain populations of trout, or whether switching to anadromy is facultative.
From each location, 4-21 samples (12.5 on average) were collected ( Figure 1). In addition, we analyzed 44 sequences of brown trout downloaded from GenBank (Table 2). Fish were fin-clipped and then returned to their natural habitats. Fin clips were dried on filter paper and stored at room temperature until DNA extraction.

| Phylogenetic analyses
We generated a mitochondrial phylogeny of the studied individuals with Bayesian inference (BI) using the software BEAST ver. 2.6.3 (Drummond et al., 2012). Settings for BEAST included: relaxed lognormal clock, Yule model, random distribution of the offspring number among the individuals; MCMC = 100,000,000; nucleotide substitution models were selected using MEGA software (Kumar et al., 2018). Additionally, we used the Median-Joining approach (Bandelt et al., 1999) for networking individual haplotypes of the specimens, with software NETWORK 10.2.0 (Bandelt et al., 1999).

| Population genetic analyses
Individual microsatellite profiles were subjected to principal component analysis (PCA), to visualize clustering of the populations in PCA space. The software used was SPSS 23.0 for Windows (IBM corp., 2015).
We used STRUCTURE ver. 2.3.4 (Pritchard et al., 2000) to evaluate the genetic structure across the Ponto-Caspian populations. In the analyses we used an admixture model, assumed independent allele frequencies, and 50,000 iterations as burn-in, with a total of 500,000 iterations. The number of potential population clusters (K) was set from 1 to 19 with five independent runs for each. The most probable K was chosen using the online tool Structure Harvester (Earl & VonHoldt, 2012).

F I G U R E 1 Sampling locations of brown trout: Colors show ultimate destination of drainage, red indicates Black Sea, blue indicates
Caspian Sea. Population 6, shown in green, indicates the location of fish whose mitochondrial DNA clustered with Salmo rizeensis (Turan et al., 2009).

TA B L E 2
The number of samples from each location and species used for the analysis of mitochondrial Cyt-b gene and microsatellite genotypes (STR) at 10 loci.  and the sum of squared differences (R ST -like), for validating the concurrence of the estimates based on these two metrics.
We used the models comparing two groups (Black and Caspian Sea basins) and six groups (population outliers from Chvana and Shareula, the rest of the populations from the Black Sea basin, populations from Kura, Tergi, and Sulak drainages).

| Inferring isolation by distance
We used simple Mantel tests (Manly, 1986) (Figures 1 and 3). AMOVA analysis results are shown in

| Geographic isolation versus genetic distances
There was significant positive correlation between the ln- rivers close to the sea was commonly smaller than the genetic distances among distant populations from within the same river basin.
In other words, isolation by distance appears to be a stronger driver of genetic differentiation than water salinity/transit through the Black Sea.

| DISCUSS ION
Addressing the first hypothesis formulated in the introduction of this paper, we suggest that the populations of brown trout from the Black and the Caspian Sea basins are genetically isolated from one another. Previously, this hypothesis was confirmed based on mitochondrial DNA analysis (Ninua et al., 2018); additional mt-DNA sequences obtained during this research also confirm this conclusion. Distinct position of these two groups reflected in PCA, the outcome of STRUCTURE clustering, and significant between-group component of genetic variation, based on the microsatellite genotype analysis, additionally confirms with this hypothesis. This result is somewhat unexpected, since brown trout is a historically popular target for fishing and breeding (see Sabaneev, 1875), and has likely been subject to artificial relocation.
The analysis of microsatellite genotypes of the sampled populations also supports isolation by distance within different sea basins.
Most of the brown trout populations are euryhaline (Berg, 1985), genotypes, exceeds the genetic distance between the trout from the Black and Caspian Sea basins. We suggest that trout from this location, along with resident fish from northeastern Turkey, belong to a distinct species, S. rizeensis. Elliot (1994) showed that resident and anadromous forms of brown trout coexist (and, probably interbreed) in many rivers draining into the Atlantic. Jonsson and Jonsson (2006) suggest that the resident versus anadromous life history may depend on the presence of physical barriers separating river habitat from the sea, or simply on the distance from the particular location to marine habitat; they also can co-occur in the same river. The resident and anadromous morphs differ in adult mass (Jonsson, 1985); age at maturity (Jonsson & Jonsson, 2006); and egg size (Olofsson & Mosegaard, 1999); Jensen et al. (2019) suggested that trade-off between fish mortality and reproductive success is a trigger of switching between the life histories. Jonsson and Jonsson (2006) also suggest the presence of genetic differences between the resident and anadromous morphs based on some differences in heritable characters, although environmental conditions can also lead to switching between the life histories. In particular, water level in a river may trigger change to and from anadromy (Jonsson et al., 2018). Findings of King et al. (2021) showed that commercially bred brown trout, stocked into a natural habitat of anadromous S. trutta, remain genetically distinct for at least 13 years after the sticking. This indirectly supports the presence of genetic differences between the non-migratory and anadromous populations of trout. The other species of the genus Salmo, Atlantic salmon (S. salar), is usually anadromous and rarely produces resident forms; however, resident forms of S. salar are landlocked and isolated from the anadromous fish, most likely, since early Holocene (Berg, 1985). There is evidence for different gene pool in resident and anadromous forms of S. salar, including a higher allelic richness in populations of the latter form and stronger genetic

TA B L E 4
The output of AMOVA analysis based on the microsatellite profiles of the studied populations.
The genetic basis of anadromy is better studied in rainbow trout (O. mykiss) from the Pacific Ocean; in this species, multiple genes are identified that influence life history; anadromy can be present or absent within the same polymorphic populations, depending upon unique constellations of alleles aggregating in individuals (Hecht et al., 2013;Le Bras et al., 2011;Nichols et al., 2008). Arostegui et al. (2019) showed that steelhead (the anadromous form of O. mykiss) is ancestral form related to the residential rainbow trout, and transition to the resident freshwater life history is secondary rather than primary in this group.
To conclude, common and independent change between the anadromous and resident life histories in trout is a multifactorial process that may occur both between conspecific populations, and also individuals of the same population, and is determined simultaneously by genetic and environmental factors.
Much less is known about the variation in life history and reproductive strategies of brown trout from the Ponto-Caspian drainages, including wide-spread S. labrax and S. caspius, than in the S. trutta from the Atlantic drainage. Sabaneev (1875) considered Black Sea salmon and brown trout from the Black Sea drainage as two different species, and only in the 20th century it was shown that they are two forms of the same species (Barach, 1962;Berg, 1959 Considering that during the glacial periods, the salinity of the Black Sea was substantially lower than now (Soulet et al., 2010) this could well happen. The questions remain-what was the trigger of separation between the two lineages, both of which could migrate through the coastal zone; and why might one of these lineages, in different parts of its range, turn from anadromous to residential life history.
The reasons for the lineage separation within Ponto-Caspian trout are discussed in Ninua et al. (2018). In fact, there are at least three almost equidistant mitochondrial lineages in the Black Sea trout, including S. rizeensis, and those lineages could emerge in case of temporary impenetrability of the Black Sea area, perhaps as a result of ice covering a large part of its surface during the periods of glaciation (Briceag et al., 2019) or alternatively during interglacials when Black Sea water was brackish and oxygen deficient due to impact of Mediterranean water inflow (Hoyle et al., 2021;Wegwerth et al., 2019). The cause for the switch to an exclusively freshwater life history in S. rizeensis remains unclear. This turns us to a question of sympatric speciation, which according to multiple recent publications is thought to be a speciation mode not less common than the "classical" pattern, wherein geographic isolation triggers early stages of speciation (Miles et al., 2018;Nunes et al., 2022).
In conclusion, our study shows that brown trout from the stud- project administration (lead); supervision (lead); writing -review and editing (supporting).

ACK N OWLED G M ENTS
The research was financed by Shota Rustaveli National Science Foundation of Georgia (award no. NFR-18-6953). We thank Mari Murtskhvaladze, Mariam Osepashvili for assistance in the laboratory and Sandro Chubinidze for assistance during sample collection. The authors appreciate Associate Editor and two anonymous reviewers for very helpful comments on the first draft of the manuscript.

CO N FLI C T O F I NTE R E S T S TATE M E NT
None declared.

DATA AVA I L A B I L I T Y S TAT E M E N T
DNA sequences: GenBank accessions OP849668-OP849680.

A PPE N D I X 3
The allocation of the studied individuals to the clusters (K) inferred with STRUCTURE algorithm (considering 1 < K < 14). For the location numbers, see Table 1.